Lipocortins are a family of proteins that have been implicated in the regulation of various aspects of inflammation [R. J. Flower et al., "Macrocortin And The Mechanism Of Action Of The Glucocorticoids", Advances in Inflammation Research, 7, pp. 61-9 (1984); M. DiRosa, "Role In Inflammation Of Glucocorticoid-Induced Phospholipase Inhibitory Proteins", Prog. Biochem. Pharmacol., 20, pp. 55-62 (1985)]. Lipocortins are believed to exert their anti-inflammatory effect by inhibiting phospholipase A.sub.2 [F. Hirata et al., "A Phospholipase A.sub.2 Inhibitory Protein In Rabbit Neutrophils Induced By Glucocorticoids" Proc. Nat. Acad. Sci USA, 77, No. 5, pp. 2533-36 (1980)]. The enzyme phospholipase A.sub.2 acts on membrane phospholipids to release arachadonic acid, a precursor in the synthesis of compounds such as prostaglandins, hydroxy-acids and leukotrienes, that are involved in the inflammatory response.
While many studies have been performed with crude protein preparations, recent studies with purified lipocortins have confirmed that these proteins are potent inhibitors of the production of mediators of inflammation [B. Rothhut et al., "Purification And Characterization Of A 32 kDa Phospholipase A.sub.2 Inhibitory Protein (Lipocortin) From Human Peripheral Blood Mononuclear Cells", FEBS Letters, 219, pp. 169-75 (1987)]. Moreover, recombinant lipocortins have been shown to inhibit the action of phospholipase in in vitro assays [G. Cirino et al., "Recombinant Human Lipocortin 1 Inhibits Thromboxane Release From Guinea-Pig Isolated Perfused Lung", Nature, 328, pp. 270-2 (1987); G. Cirino and R. J. Flower, "Human Recombinant Lipocortin 1 Inhibits Prostacyclin Production By Human Umbilical Artery In Vitro" Prostaglandins, 34, pp. 59-62 (1987)] and in an in vivo model of the inflammatory response [G. Cirino et al., "Human Recombinant Lipocortin I Has Acutal Local Anti-Inflammatory Properties In The Rat Paw Edema Test", Proc. Nat. Acad. Sci. U.S.A., 86, pp. 3428-32 (1989)].
As a result of their demonstrated anti-inflammatory action, lipocortins should prove useful in the treatment of disorders characterized by inflammatory processes. Examples include arthritic, allergic, dermatologic, ophthalmic and collagen diseases. Furthermore, the use of lipocortins may eliminate side effects associated with currently available anti-inflammatory treatments.
To date, related lipocortin proteins with molecular weights of about 70, 55, 40, 30 and 15 kd have been detected in a variety of animal tissues. K-S Huang et al., "Two Human 35 kd Inhibitors Of Phospholipase A.sub.2 Are Related To Substrates Of pp60v-src And Of The EGF Receptor/Kinase", Cell, 46, pp. 191-99 (1986). However lack of structural studies has hampered the precise delineation of the family of lipocortins.
Recombinant DNA technology holds significant promise for progress in this field. In the first place, it will help to define the scope of the family of lipocortins, by allowing elucidation of the primary structure of the member proteins. Second, it is desirable to be able to prepare large quantities of lipocortins using recombinant DNA technology. Although it is possible to purify lipocortins from biological sources, production by recombinant methods is more economical. In addition, recombinant methods allow large scale synthesis of desired fragments or otherwise modified versions of native lipocortins.
Two lipocortin genes have been cloned previously [Wallner et al., "Cloning And Expression Of Human Lipocortin, A Phospholipase A.sub.2 Inhibitor With Potential Anti-Inflammatory Activity", Nature, 320, pp. 77-80, 1986; Huang et al. 1986; C. J. M. Saris et al, "The Sequence Of The cDNA For The Protein Kinase Substrate, p36, Reveals a Multidomain Protein With Internal Repeats", Cell, 46, pp. 201-12 (1986)]. Both encode homologous 38 kd proteins with 50% amino acid homology. These two proteins have been termed lipocortin-I and lipocortin-II by Huang et al., 1986.